Abstract Assessing whether myeloid cells support HIV-1 persistence in the face of effective ART represents a significant technical challenge. As a result, there is as yet, no direct evidence that myeloid cells play any role in viral persistence in the face of effective ART. As a consequence, research on myeloid cell reservoirs is falling into obscurity. Infection of macrophages can only be initiated by HIV-1 variants that have the ability to use low levels of CD4 on the cell surface. Therefore, if a functional myeloid reservoir contributes to viral persistence under effective ART, we would predict that viremia that rebounds following analytic treatment interruption (ATI), would contain viral variants that have a high affinity for CD4. We have developed an approach that allows identification of low frequency macrophage-tropic variants in rebounding viremia post ATI. Through single genome amplification (SGA), we cloned a large number of viral envelopes from plasma of individuals who underwent ATI. When these envelopes were used to construct recombinant molecular clones, they conferred the ability to fuse with, and replicate within primary macrophages. We believe that these results provide definitive evidence for the existence of a myeloid cell reservoir in infected individuals on suppressive ART and furthermore, that this reservoir contributes to viral rebound when ART is interrupted. We hypothesize that the myeloid cell reservoir is stable, permits long term viral persistence under suppressive ART and fuels viral rebound upon treatment interruption. To pursue this hypothesis, we propose to: 1: Assess the frequency of macrophage-tropic viruses through longitudinal sampling of rebounding viremia post-ATI. 2: Assess the persistent nature of the myeloid cell reservoir from individuals with prolonged remission intervals following bone marrow transplant (Boston patients) as well as sampling of rebounding viremia from individuals who underwent sequential ATI. 3: Evaluate whether macrophage-tropic viruses have a CNS origin and whether they are less fit relative to T tropic viruses. This proposal will integrate clinical, virologic and molecular studies to reveal the existence of a myeloid cell reservoir in individuals on suppressive ART and to shed light on the dynamics of this reservoir. The critical information gained from this study will provide whether there is rationale for an expanded effort to develop strategies to target the myeloid cell reservoir and to accelerate the path to a viral cure.